Reduction of Tobacco-Specific Nitrosamines Using Genetic Modification to Elevate Production of Native Antioxidants in Tobacco

ABSTRACT

Cured tobacco having a lower concentration of tobacco specific nitrosamines, can be produced from a transgenic tobacco plant having a heterologous nucleotide sequence encoding a polypeptide that catalyzes production of active oxygen species. The heterologous nucleotide sequence is operably coupled to a regulatory nucleotide sequence that directs expression of the polypeptide. The regulatory nucleotide sequence can include constitutive and/or inducible promoter elements.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to methods of producing tobacco having reducedlevels of tobacco specific nitrosamines in the cured leaves andtransgenic tobacco plants for producing tobacco having reduced levels oftobacco specific nitrosamines in the cured leaves.

Description of the Related Art

In many organisms, antioxidant activity increases upon exposure toconditions that increase the formation of active oxygen species such ashydrogen peroxide. There is evidence that active oxygen species areubiquitous messengers in plants that stimulate defense responses againststressful conditions.

It has been reported that tobacco specific nitrosamines (TSNAs) areproduced primarily in the curing process. It is believed thatantioxidants interfere with the nitrosation of secondary alkaloidsthereby reducing the formation of TSNAs. Commonly owned U.S. applicationSer. No. 10/235,636 discloses a method for reducing tobacco specificnitrosamine content in cured tobacco by increasing the levels ofantioxidants in the tobacco prior to harvesting. The methods disclosedin that application include root pruning prior to harvesting, severingthe xylem tissue prior to harvesting, and administering antioxidants orchemicals that produce an increase in antioxidants to the tobacco plantafter harvesting. U.S. application Ser. No. 10/235,636 and allpublications referred to below are hereby incorporated into the presentapplication entirely and for all purposes.

U.S. Pat. No. 5,516,671 discloses that plants, for example potatoes, canbe transformed with a gene encoding glucose oxidase to produce plantsthat are resistant to disease. See also, Wu et al. (Plant Physiology,115:427-435, 1997).

Berna and Bernier (Plant Molecular Biology, 33:417-29, 1997) have shownthat wheat germin has oxalate oxidase (OxO) activity and (PlantMolecular Biology, 39:539-49, 1999) that the promoter of the gemingf-2.8 gene is active in transgenic tobacco. Indeed, most germins andmany germin-like proteins have OxO activity. (Bernier and Bema, PlantPhysiology and Biochemistry, 39:545-9, 2001). PCT ApplicationPublication Number WO 99/04013 discloses the expression of oxidaseenzymes as a method of producing resistance to stress in a variety ofplants.

Published U.S. Application No. 2004/007218 discloses a method fordetermination of the level of H₂O₂ in a cell by measuring expression ofa reporter polypeptide from a promoter that is inducible by H₂O₂.

SUMMARY

By supplying a source of active oxygen species, for example hydrogenperoxide, that is independent of stress and/or that amplifies a responseto stress, increased concentrations of antioxidant molecules can beinduced in tobacco thereby providing for lower levels of tobaccospecific nitrosamines in the cured tobacco.

We describe cured tobacco having reduced levels of tobacco specificnitrosamines, which is produced from a transgenic tobacco plantcomprising a heterologous nucleotide sequence encoding a polypeptidethat catalyzes production of active oxygen species. Of course, theheterologous nucleotide sequence is operably coupled to a regulatorynucleotide sequence that directs expression of the polypeptide. Inpreferred embodiments, the polypeptide that catalyzes production ofactive oxygen species is an oxidase, for example an oxidase selectedfrom among oxalate oxidase and glucose oxidase. In various exemplaryembodiments, the regulatory nucleotide sequences can comprise aconstitutively active promoter, an inducible promoter, or elements thatdirect a level of constitutive expression of the polypeptide andinducible increased levels of expression. In preferred embodiments, thepromoter is an inducible promoter or comprises a promoter element can beinduced by treating the plant with an inducer, which can be physicaland/or chemical. For example, various known plant promoters can beinduced by a plant wounding treatment (e.g. root pruning or cutting thexylem tissue), chemical inducers, heat, light, drying, combinations ofthe foregoing, and the like.

A method of producing cured tobacco having reduced concentrations oftobacco-specific nitrosamines is disclosed. In preferred embodiments,the method comprises growing a transgenic tobacco plant having aheterologous nucleotide sequence encoding a polypeptide that catalyzesproduction of active oxygen species, for example as described above. Theheterologous nucleotide sequence is coupled to a regulatory nucleotidesequence that directs expression of the polypeptide. The transgenictobacco is harvested and cured.

In preferred variations of the method, the transgenic tobacco plantcomprises an inducible promoter or inducible promoter elements operablycoupled to the heterologous nucleotide sequence encoding a polypeptidethat catalyzes production of active oxygen species. In some variants ofthe method, the promoter may be arranged to produce a level ofconstitutive expression of the polypeptide and a higher level ofexpression in response to an inducer. Where the promoter is an induciblepromoter or comprises inducible promoter elements, the method preferablyfurther comprises treating the tobacco to induce expression of thepolypeptide prior to or at about the time of harvest. For example thetobacco may be treated with an inducer sufficient to induce expressionof the polypeptide about 1 week or less prior to harvest, between about1 to 3 weeks prior to harvest, or more than about 3 weeks prior toharvest. Alternatively, or in addition, the tobacco may be treated withan inducer at the time of harvest or within about 48 hours afterharvest. In some embodiments, the natural process of senescence or thephysical process of harvesting may comprise the inducer so that furthertreatment of the plant is unnecessary to induce expression.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cured tobacco that has lower levels of tobacco specific nitrosamines(TSNAs) can be produced from a transgenic tobacco plant that comprises aheterologous nucleotide sequence encoding a polypeptide that catalyzesproduction of active oxygen species where the heterologous nucleotidesequence is operably coupled to a regulatory nucleotide sequence thatdirects expression of the polypeptide.

Without wishing to be bound by theory, it is believed that theproduction of active oxygen species triggers defense responses in thetobacco plant that include the production of antioxidants. It isbelieved that such antioxidants interfere with the nitrosation ofsecondary alkaloids thereby reducing the formation of TSNAs. Preferredtobacco varieties of tobacco plants include Burley, Oriental, andbright. Burley is a most preferred variety.

Active oxygen species include, for example, peroxides such as hydrogenperoxide (H₂O₂), O₂ ⁻, and OH⁺. In preferred embodiments, thepolypeptide that catalyzes production of active oxygen species is anoxidase, for example an oxidase selected from among oxalate oxidase andglucose oxidase. Other polypeptides, including proteins, which catalyzethe production of active oxygen species are known to those skilled inthe art, for example, acyl coA oxidase, aspartate oxidase, cholineoxidase, copper amine oxidase, eosinophil peroxidase, flavin oxidase,galactose oxidase, glycolate oxidase, monoamine oxidase, polyamineoxidase, NADPH oxidase, xanthine oxidase, and the like.

Preferred oxidase enzymes include gerrnin-like oxalate oxidase andglucose oxidase. Oxolate oxidase (OxO) catalyzes the degradation ofoxalic acid into H₂O₂ and CO₂. The coding sequence of a germin-like OxOgene was isolated and genetically engineered for constitutive expressionin plants by Bernier and Berna (Plant Physiology and Biochemistry,39:545-9, 2001). Wu et al. (Plant Physiology, 115:427-435, 1997) createda transgenic potato plant expressing a fungal glucose oxidase gene thatdemonstrates some resistance to pathogens. The level of accumulation ofsalicylic acid in the leaves of the potato plant increased and theproduction mRNA's of defense related genes encoding anionic peroxidaseand chitins were also induced. Constitutively elevated levels of H₂O₂appear to activate an array of host defense mechanisms including theproduction of antioxidants.

As used herein, heterologous nucleotide sequence means a nucleotidesequence, such as a gene sequence or the coding sequence of a gene,which is derived from a different organism than the host organism inwhich it has been placed and/or a nucleotide sequence, which may includea sequence native to the host organism, that has been cloned from itsnative location and manipulated so as to be coupled with sequence withwhich it is not naturally coupled. For example a sequence encoding anative protein may be coupled to a non-native promoter sequence, anative promoter sequence can be coupled to a non-native protein, ornative protein and promoter sequences that are naturally found indifferent genes may be couple and reintroduced into a host organism. Atransgenic plant is a plant having a heterologous nucleotide sequence inits cells. Reliable methods for cloning a heterologous nucleotidesequence and introducing the heterologous gene into plant cells so as toproduce a transgenic plant are well known to the skilled practitioner.

Regulatory sequences include those sequences necessary for transcriptionand/or translation of a coding sequence. For example, regulatorysequences of a gene generally include a promoter sequence. Promoters canbe constitutively active, providing for continuous expression of a gene,or may be inducible, providing for expression of a gene in response toan inducer stimulus. A promoter may also comprise elements that providefor a level of constitutive expression coupled to elements that providea higher level of expression in response to one or more inducers.

A preferred type of promoter is a tissue specific promoter, which canprovide for a tissue specific expression profile, for example whereexpression is greatest in the roots and stems of a plant. Exemplarytypes of inducible promoter include stress response promoters, lightinducible promoters, and chemically inducible promoters, such aspromoters that are inducible by active oxygen species or another stressresponse product and/or by compounds produced in response to high lightexposure and/or by synthetic compounds. Using a stress response promoterto express a heterologus source of active oxygen species can provideamplification of a stress response to a physical or chemical treatment.

In preferred embodiments, the regulatory nucleotide sequences cancomprise a constitutively active promoter, an inducible promoter, orelements that direct a level of constitutive expression of thepolypeptide and inducible increased levels of expression. In exemplaryembodiments, the inducible promoter or promoter elements can be inducedby plant wounding treatment, for example by root pruning or cutting thexylem tissue of a tobacco plant. In alternative embodiments, theinducible promoter can be induced by treatments such as chemical, heat,light, drought, combinations thereof, and other treatments chosenaccording to the known properties of the regulatory sequence.

If desired, a tissue specific promoter may be used to direct expressionof the polypeptide to a chosen part of the tobacco plant. For example, aroot specific promoter can be used to direct greater expression of thepolypeptide in the roots. A preferred example of a tissue specificpromoter includes the Figwort mosaic virus (FMV) sub-genomic transcript(Sgt) promoter and various modified varieties of the FMV Sgt promoter,such as the fragment of the FMV Sgt promoter comprising from aboutposition −270 to about +31. This fragment was shown to be about twotimes stronger than the CMV 35S promoter in tobacco and to demonstrate atissue specific expression in tobacco root that is greater than in leafand stem. (Bhattacharyya et al., Virus Research, 90:47-62, 2002). Thecomplete FMV genome can be found in the NCBI database under accessionno. NC_ 003554 . Other FMV promoter sequences are disclosed in U.S. Pat.Nos. 6,018,100 and 5,378,619.

If desired, expression of the polypeptide encoded by the heterologousnucleotide sequence can be regulated by a promoter that responds tonatural signals associated with senescence. For example, a promoter maybe chosen from among the promoters of senescence-associated genes(SAGs). Many such genes have been identified. (Gepstein et al., ThePlant Journal, 36:629-42, 2003). Temporal patterns of gene expressionduring senescence have been observed. Thus, gene promoters can be chosenthat have a level of constitutive activity, but are strongly upregulatedduring senescence. Among this group are promoters driving expression ofcationic amino acid transporters, amino acid permease, andmetallothionin. Promoters can be chosen from SAGs that are upregulatedearly in senescence. Among this group are promoters of the RING-H2finger protein and the promoter of the xylose isomerase gene.Alternatively, a promoter can be chosen that is upregulated in the latestages of senescence. Among this type of promoter is the lethal leafspot 1 (lls1) gene promoter. The use of a SAG promoter as a regulatorynucleotide sequence can permit timing the expression of the heterologousnucleotide sequence to coincide with natural events or treatments thatprecede harvest by a desired period. Alternatively, or in addition,because certain SAG promoters can be strongly upregulated by stressevents such as leaf detachment, the use of selected SAG promoters canprovide for upregulation of expression due to natural or artificialstressors and/or at the time the tobacco is harvested. When a SAGpromoter is used, it may be unnecessary to further treat transgenictobacco to obtain expression of a polypeptide that catalyzes productionof active oxygen species for a desired period prior to and/or at thetime of harvest.

If desired, a chemically inducible promoter can be chosen to permitinduction of expression of the polypeptide in response to a non-naturalsignal. The regulatory sequence can comprise recognition elements thatbind an engineered transcriptional activator. And the transgenic tobaccomay be designed to produce a ligand binding transcriptional activator.For example, a glucocorticoid-inducible transcriptional activator (GVG)can be constructed comprising a Ga14 binding domain, a VP16 activationdomain, and a gluccocorticoid receptor. Expression from a GVG activatedpromoter can be induced using micromolar concentrations of dexamethasone(DEX). (Ouwerkerk et al., Planta, 213:370-8, 2001). Examples of binaryvectors using DEX inducible promoters can be found in GenBank underaccession nos. AF294979-AF294982. As further examples, synthetictranscriptional activators comprising the ligand binding domain of theecdysone receptor from spruce budworm, a VP16 activation domain and GAL4and LexA DNA binding domains can be used to respond to methoxyfenozideto induce a heterologous nucleotide sequence operable coupled to a Ga14or LexA-response element in a synthetic promoter. Alternatively, nativeinducible promoter elements can be coupled to the heterologousnucleotide sequence to drive inducible expression. An inductiontreatment could be performed by spraying or irrigation. The use ofchemically inducible promoters, including field use with induction byregistered agrochemicals, has been reviewed by Zuo (Current Opinion inBiotechnology, 11:146-51, 2000).

Promoters that can be used as nucleotide regulatory sequences includesynthetic plant promoters comprising a plurality of cis-acting elements.For example, various responsive elements can be inserted upstream of aminimal promoter segment such as a minimal CMV 35S promoter segment.(Rushton et al., The Plant Cell, 14:749-62, 2002). Tandem repeats may beused to increase the response of a promoter. Additional copies ofelements can increase constitutive expression in addition to aninducible response. Such a synthetic promoter can also be designed tohave tissue specificity, such as a preference for expression in rootover expression in leaf.

A wide variety of additional suitable regulatory sequence elements andtheir properties are known to those of skill in the art. For example,the cauliflower mosaic virus 35S promoter is commonly used forconstitutive expression. The arabidopsis FAD7 promoter provides bothconstitutive expression and wound induced expression. Wound responsiveelements of the FAD7 promoter have been characterized. (Nishiuichi etal., Plant Physiology, 121-1239-46, 1999). Thus, a modified FAD7promoter can be constructed comprising all or part of the FAD7 promoter.When using such a promoter, induction may be by wounding or by providinga chemical source of the wound response messengers that can directlyinduce the promoter. The hydroxyproline-rich glycoprotein (HRGP4.1)promoter from bean demonstrates a tissue specific expression profile ofreporter gene that is 7-fold higher in root than stem and 10-fold higherin stem than in leaves of transgenic tobacco. The HRGP4.1 promoter alsoexhibits a stress response that provides for localized induction inresponse to wounding. (Wycoff et al., Plant Physiology, 109:41-42,1995). The KatA promoter of Agrobaterium tumefacines is an example of apromoter that is inducible by active oxygen species such as H₂O₂. QuanPan, Published U.S. Patent Application No. 2004/0072218. The Early-LightInducible Protein 2 (ELIP2) promoter is induced by messengers triggeredby strong light exposure in a pathway that is independent of hydrogenperoxide. (Kimura et al., Photochem Photobiol. 77:668-74, 2003). CMA5 isa native 52-bp fragment of the Nicotiana plumbaginifolia rbcS 8Bpromoter, which contains an I- and a G-box cis-element. CMA5 behaves asa light-responsive minimal unit capable of activating a heterologousminimal promoter in a phytochrome-, cryptochrome-, and plastid-dependentmanner. (Martinez-Hernandez et al., Plant Physiology, 128:1223-33,2002). These examples are illustrative of the various types promotersthat may be chosen, but should not be considered limiting.

Expression driven by inducible promoters can be induced according to thecharacteristics of the chosen promoter either directly by introductionof chemical messengers or by various treatments including wounding,heat, light, drought, combinations thereof, and the like. One skilled inthe art is capable of selecting and operably coupling appropriateregulatory sequence elements to a coding sequence to provide inductionof polypeptide expression in response to a chosen stimulus.

A method of producing cured tobacco having reduced concentrations oftobacco-specific nitrosamines can comprise growing a transgenic tobaccoplant, preferably having one or a combination of the preferredcharacteristics described above.

When the regulatory nucleotide sequence of the heterologous nucleotidesequence comprises an inducible promoter, or inducible promoter elementscoupled to additional promoter elements, the method can preferablycomprise causing induction of the promoter prior to harvest, or atharvest. In preferred embodiments, this permits the tobacco plant togrow without strong constitutive expression of stress response geneswhile providing for controlled induction of a strong response at adesired time. It may also be desirable to have a level of constitutiveexpression prior to induction. Induction may be accomplished by anytreatment consistent with the nature of the chosen inducible promoter.Induction treatment can be performed prior to harvest, for example morethan about 3 weeks prior to harvest, in the interval about 1 to 3 weeksprior to harvest, or less than about 3 weeks prior to harvest, such asabout 48 hours prior to harvest. As described above, no separateinduction treatment may be needed where the transgenic tobacco comprisesa promoter that responds to natural senescence signals. Optionally, theact of harvesting may provide the inducing stimulus, or recentlyharvested tobacco may be treated to induce the promoter, for exampletobacco may be treated with an appropriate inducer within about 48 hoursof harvest.

Harvesting and curing of the tobacco may be conducted according toconventional methods. Curing is most preferably done by air curing.Conventional air-curing tobacco barns typically utilize naturalconvection, with air flow generally proceeding from the bottom of thebarn toward the top of the barn. In curing tobacco by the proceduregenerally referred to as the “bulk curing” method, tobacco leaves aretypically loaded in a relatively compact mass on racks or in containersand placed inside of an enclosed curing barn where a furnace or aplurality of heaters circulate a forced flow of heated air through themass of tobacco leaves to effect curing and drying. Conventional tobaccocuring barns attempt to obtain the desired atmospheric conditions suchas temperature and humidity within the tobacco barn by variousadjustments of louvers or openings in the sides of the barn and theoperation of heaters spaced along the floor of the barn with respect tothe prevailing temperature and moisture content of the outsideatmosphere, the wind velocity and its direction with respect to thetobacco barn.

It may be desirable to utilize a method for curing tobacco as describedin commonly owned Published U.S. Patent Application No. 2003/0145867,which is incorporated by reference herein in its entirety. Briefly, sucha preferred method for air curing tobacco includes the tobacco beinghung in an enclosure having one or more vertically arranged air ductspositioned near the middle of the enclosure, one or more in-line fanspositioned in the air ducts, one or more ventilating fans located in anupper part of the enclosure and at least one closeable opening in a sidewall of the enclosure. The method includes opening at least one opening,and operating at least one ventilating fan to force air down through thetobacco from the upper portion of the enclosure. The method of curingtobacco can include the steps of closing an opening, introducing anaqueous solution or steam into a lower portion of a vertically arrangedair duct, and operating fans to diffuse the moisture upwards through theair duct.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention.

1. Cured tobacco having lower concentrations of tobacco specificnitrosamines, which is produced from a transgenic tobacco plantcomprising a heterologous nucleotide sequence encoding a polypeptidethat catalyzes production of active oxygen species, wherein theheterologous nucleotide sequence is operably coupled to a regulatorynucleotide sequence that directs expression of the polypeptide.
 2. Thetobacco of claim 1, wherein the polypeptide that catalyzes production ofactive oxygen species is an oxidase.
 3. The tobacco of claim 2, whereinthe polypeptide that catalyzes production of active oxygen species is anoxidase selected from among oxalate oxidase and glucose oxidase.
 4. Thetobacco of claim 1, wherein the regulatory nucleotide sequence comprisesa constitutively active promoter sequence.
 5. The tobacco of claim 1,wherein the regulatory nucleotide sequence comprises an induciblepromoter sequence.
 6. The tobacco of claim 1, wherein the regulatorynucleotide sequence comprises a promoter sequence that is induced byplant wounding, heat, light, drought, or a combination thereof.
 7. Thetobacco of claim 5, wherein the promoter is induced by a natural signalassociated with senescence.
 8. The tobacco of claim 5, wherein thepromoter is inducible by a synthetic chemical.
 9. The tobacco of claim1, wherein the regulatory nucleotide sequence comprises a promotersequence that is most active in root and/or stem tissue. 10-12.(canceled)
 13. A method of producing cured tobacco having reducedconcentrations of tobacco-specific nitrosamines, the method comprising:growing a transgenic tobacco plant comprising a heterologous nucleotidesequence encoding a polypeptide that catalyzes production of activeoxygen species, wherein the heterologous nucleotide sequence is operablycoupled to a regulatory nucleotide sequence that directs expression ofthe polypeptide; harvesting the tobacco; and curing the tobacco.
 14. Themethod of claim 13, wherein the polypeptide that catalyzes production ofactive oxygen species is an oxidase.
 15. The method of claim 13, whereinthe polypeptide that catalyzes production of active oxygen species is anoxidase selected from among oxalate oxidase and glucose oxidase.
 16. Themethod of claim 13, wherein the regulatory nucleotide sequence comprisesa constitutively active promoter sequence.
 17. The method of claim 13,wherein the regulatory nucleotide sequence comprises an induciblepromoter.
 18. The method of claim 17, comprising causing induction ofthe promoter within the period about 3 weeks prior to harvesting thetobacco to about 48 hours after harvesting the tobacco.
 19. The methodof claim 17, wherein the regulatory nucleotide sequence comprises apromoter sequence that is induced by chemical treatment, plant wounding,heat, light, drought, or a combination thereof.
 20. The method of claim17, comprising causing induction of the promoter about 1 to 3 weeksprior to harvesting the tobacco.
 21. The method of claim 17, wherein thepromoter is induced by one or more natural signals associated withsenescence.
 22. The method of claim 19, further comprising root pruningand/or severing the xylem tissue of the tobacco plant prior toharvesting.
 23. The method of claim 19, wherein the promoter isinducible by chemical treatment and further comprising applying aninducer by spraying or irrigation. 24-29. (canceled)